Abstract We developed a synthetic polymer matrix substrate (PMEDSAH) that supports induced pluripotent stem (iPS) cell and human embryonic stem (hES) cell expansion in an undifferentiated state (self-renewal) in defined culture conditions that are free from xenogeneic contamination. We have thus overcome the undefined growth conditions that typically depend on the support of mouse embryonic feeder cells (MEFs) or an undefined matrix such as MatrigelTM and that until now have severely limited our ability to perform unimpeded mechanistic studies and hindered our ability to use these stem cells to treat debilitating human diseases. Our goals for this proposal are to define the molecular mechanisms that maintain iPS cells in an undifferentiated state, the derivation of iPS cell lines in our xeno-free and fully defined culture system, and the controlled differentiation of these cells towards a mesenchymal stem cell (MSC) phenotype. We will take full advantage of our unique, xeno-free and fully defined culture system, which consists of PMEDSAH as the substrate and serum-free, defined culture medium to elucidate the mechanisms responsible for self-renewal on this substrate. Accomplishing these goals is an important prerequisite to the development of therapeutic protocols using pluripotent stem cells to regenerate human tissues. The projects outlined in this competing renewal proposal are designed to directly address these goals and our success should have a significant impact in methods to regenerate human tissues using pluripotent stem cells. It is well recognize that the microenvironment influences the fate of stem cells, thus in Specific Aim 1 we will define the structural and/or physico-chemical properties of PMEDSAH that lead to iPS cell self-renewal and maintenance of the undifferentiated state. In Specific Aim 2 we will determine the cell receptor mechanisms that direct adhesion and maintain iPS cells in an undifferentiated state on synthetic polymer substrates, testing the hypothesis that pluripotent iPS cells use more than one cell adhesion system to adhere to and support self-renewal on a defined polymer substrate. In Specific Aim 3 we will demonstrate that patient-specific iPS cells can be derived on defined substrates free of xenogeneic contamination and are able to differentiate into mesenchymal stem cells capable of regenerating craniofacial skeletal defects, as a proof of concept that our system has the potential of getting the medical and scientific community closer to clinical-grade pluripotent stem cells. Our approach is unique and fundamentally different than the state of the art because it uses synthetic components as the structural motifs in cell-substrate interactions. By accomplishing our goal, we will make significant contributions to the understanding one of the major unresolved issues in pluripotent stem cell biology; that is, learning how pluripotent stem cells interact with their extracellular environment to: 1) remain in a unique undifferentiated state and 2) make fate changing lineage decisions. This knowledge is important for both understanding basic stem cell biology and developing consistent and safe regenerative therapies.